Many types of human cancer are now believed to be caused by an imbalance of growth regulators within a cell. A decrease in negative control growth regulators and/or their deactivation can cause a cancerous condition. Further, an increase in positive control growth regulators can also cause a cancerous condition.
Since the identification of the first tumor suppressor gene, much effort in cancer research has been focused on the identification of cellular proteins which interact with tumor suppressor proteins and their involvement in human cancer. Moreover, many types of human cancers are thought to develop via mechanisms which impair the function of tumor suppressor genes.
One of the most studied tumor suppressor genes is the retinoblastoma susceptibility gene (RB), whose gene product (pRb) has been shown to play a key role in the regulation of cell division. R. A. Weinberg, Science, 254:1138 (1991). pRb is a nuclear protein that acts as a cell cycle control checkpoint by inhibiting G1/S progression. In interphasic cells, pRb contributes to maintaining the quiescent state of the cell by repressing transcription of genes required for the cell cycle through interaction with transcription factors, such as E2F (see e.g. Hiebert et al., Genes Develop., 6, 177-185 (1992)). Upon entrance into the cell cycle, pRb is phosphorylated by cell cycle-dependent kinases (see e.g. Matsushime et al., Nature, 35, 295-300 (1992)) which is thought to permit its dissociation from transcription factors and, hence, the expression of genes required for progression through the cell cycle. The association of pRb with cell cycle regulators like cyclins and cell cycle-dependent kinases suggests a universal character to its function.
Deletion or inactivation of both RB alleles is an essential, rate-limiting step in the formation of retinoblastoma and osteosarcoma that arise within families that carry a mutated RB gene. RB inactivation is also found in other sarcomas, small cell carcinoma of the lung, and in carcinoma of the breast, prostate and bladder. The restriction of pRb's involvement in human cancer to a limited number of tumor types suggests that its hypothetical universal function is influenced by other gene products in a cell type-specific manner. Consistently, knock out of the RB gene in mice affects only specific cell types after several days of embryonic development (see e.g. Clarke et al., Nature, 359, 328-330 (1992)). The loss of this activity can induce cell transformation as evidenced by the reversion of the transformed phenotype in pRb cells after replacement of a functional pRb. Moreover, injection of the RB gene product, pRb, into G1 cells can block cell cycle progression. Thus the identification of factors that interfere with and/or control pRb function is critical for understanding both cell cycle control and oncogenesis.
A major advance in the search for pRb function was the finding that pRb is a target for oncogenic products of DNA tumor viruses. Initial studies demonstrated that the adenovirus E 1A protein forms a complex with Rb which is dependent on sequences in the E1A protein. P. Whyte, et al., Nature, 334:124 (1988). Certain transforming proteins such as SV40T and E7, which are derived from the transforming or tumor-associated subtypes of adenoviruses and human papilloma viruses (HPV) are also capable of binding to pRb, thereby blocking its normal function. All these viral oncoproteins interact with pRb through an LXCXE motif (where X is any amino acid). Interaction of these viral proteins with pRb appears to be an important aspect of their oncogenic potential in which an inactivation of pRb function is achieved, that is equivalent to a deletion or mutation of RB. Growth factors such as transforming growth factor-β1 (TGF-β1) also exert their cell cycle control via pRb. A. B. Roberts, et al., Peptide Growth Factors And Their Receptors, 421-427 (1990). The mechanism(s) by which TGF-β1 inhibits cellular proliferation involves an attenuation of phosphorylation of pRb at the G1/S transition of the cell cycle. See e.g. I. Reynisdottir, et al. Genes Dev., 9:1831 (1995).
The ability of several transforming proteins from human DNA tumor viruses to activate cell proliferation has been a useful tool for the identification of cellular factors involved in the regulation of the cell cycle. Negative regulators of cell growth may thus be effective targets for inactivation by these viral proteins, as it occurs with the product of the retinoblastoma gene. Adenovirus E1A, SV40 T antigen, and papillomarivus E7 are three exemplary viral proteins which have been found to bind to pRb. This binding is responsible for the release of transcription factors required for the expression of cell cycle genes (see e.g. Nevins, Science. 258, 424-429 (1992).
A conserved motif found in the three viral proteins allows for interaction and complex formation with pRb (Moran, Curr. Op. Gen. Dev., 3, 63-70 (1993)). In the case of the adenovirus E1A protein, this motif is located in the transforming domain 2, which is required for growth activation. The pRb-related product p107 also binds in this region. Domain 2 is also the site of interaction of an additional E1A-binding protein, p130. This has led to the suggestion that p130 has a structural relationship to pRb and p107 (Moran, Curr. Op. Gen. Dev., 3, 63-70 (1993)).
The viral oncoprotein-binding domain in pRb, p107 and p130 is a conserved region termed the “pocket region” (see e.g. Ewin et al., Cell, 66, 1155-1164 (1991)), and it is thought to play a primary role in the function of these proteins. The pocket is structurally formed by two regions A and B, which are conserved in pRb, p107 and p130 and separated by nonconserved spacers of different sizes in pRb, p107 and p130.
In addition to its interaction with viral oncoproteins, pRb is known to bind at least two dozen cellular proteins. J. Wang, Curr Opin Gen. Dev, 7:39 (1997); Y. Taya, et al., Trend Biochem. Sci., 22:14 (1997). Included in these is the transcriptional factor E2F-1 which is required for the transcription of certain cellular genes that participate in growth control and DNA synthesis (see e.g. W. B. La Thangue, et al., Biochem. Soc. Trans., 24:54 (1996). It is now clear that E2F is composed of a family of closely related proteins, E2F-1 to -5, and a set of partner proteins belonging to the DP family of transcriptional factors (see e.g. J. E. Slansky, et al., Curr. Top. Microbiol. Immunol., 208:1 (1996). E2F binds preferentially to the hypophosphorylated form of pRb and to several Rb related proteins, including p107 and p130. E2F can be dissociated from pRb and related protein complexes by two mechanisms: hyperphosphoylation of pRb or by the competitive binding with viral proteins. Treatment of cells with TGF-β1 results in the accumulation of pRb in the hypophosphorylated form. Hypophosphorylated pRb binds E2F-1 thus blocking the growth promoting activity of free E2F-1. However, co-expression of viral pRb binding proteins can reverse the TGF-β1 mediated arrest of cell growth presumably by displacing E2F-1 from pRb. K. Smith, et al. Virology, 224:184 (1996); K. Zerfass. J. Gen. Virol. 76:1815 (1995). Thus, hypophosphorylated pRb binds E2F-1 thereby blocking E2F-1 growth promoting activity whereas phosphorylation of pRb or complexing with viral oncoproteins releases E2F from pRb and leads to transcriptional activation of growth promoting genes.
The association of pRb with transcription factors, such as E2F, occurs by interactions at the pocket region and, recently, p107 has also been shown to exert such a binding profile. Moreover, the pocket region is found mutated in several human cancers where a lack of function of the pRb protein is thought to be involved in the acquisition of the transformed phenotype.
There is a need for identification and characterization of new cellular genes encoding proteins which interact with pRb and which may be involved in the regulation of cell growth. The isolation and characterization of genes encoding proteins which interact with pRb are particularly useful in studying mechanisms of cell proliferation and the means to modulate such activity.